Polyketides are a large family of structurally diverse natural products with a broad range of biological activities. These polyketides, although structurally complex and rich in stereochemistry, are elegantly synthesized in biological systems via a predictable modular polyketide synthases (PKSs) paradigm. The modular nature of PKSs presents significant opportunities for the engineering of novel compounds. However, the rational exploitation of the functional modularity of PKSs is currently limited by an incomplete understanding of the biochemical reactions involved in the polyketide assembly. The goal of the proposed research is to identify the kinetic bottlenecks that limit the processing of a growing polyketide through hybrid PKSs. We propose to achieve this goal through the following Specific Aims: i) Construct a dissociated bimodular system that represents modules 1 and 3 of 6-deoxyerythronolide B synthase (DEBS);ii) Determine the reaction kinetics of the entire dissociated bimodular system and each distinct biochemical reaction to identify the rate-limiting steps in polyketide processing;ill) Evaluate the rate-liming biochemical reactions in other dissociated bimodular hybrid PKSs to ascertain if the same steps in polyketide processing create a bottleneck in other chimeric systems. These goals will be achieved by constructing acyl carrier protein domains (ACP) and keto synthase - acyl transferase didomains ([KS][AT]) from the DEBS PKS to build a dissociated hybrid PKS. Within this system, the kinetics of domain acylation and ketide-unit elongation will be studied as discrete catalytic events by radio-SDS PAGE and radio-TLC. The proposed research will identify the current limitations in harnessing chimeric PKSs for the production of polyketides. We anticipate that the results of the proposed study will be broadly applicable to chemical engineering by improving the current understanding of the fundamental principles that govern polyketide antibiotic biosynthesis. Ultimately, we expect our findings to contribute important tools for the rational engineering of novel polyketides with potential antibiotic, anti-tumor, and other pharmacologically relevant biological activities. PUBLIC HEALTH RELEVANCE: Insights from the proposed mechanistic study will be applied to identify and efficiently produce novel polyketides with pharmacologically relevant biological activities.